This application addresses the broad Challenge Area (03) Biomarker Discovery and Validation, and the specific Challenge Topic, 03-AR-101: Biomarkers of Persistent Damage after Acute Joint Injury. Serum microRNAs as biomarkers of post-traumatic osteoarthritis. This study proposes to define a panel of serum miRNAs and validate their use as biomarkers of post- traumatic osteoarthritis (PTOA). Approximately 12% or 5.6 million patients suffering osteoarthritis (OA) in the United States can attribute their disease to prior joint injury. However;since not all joint injuries progress to joint degeneration there is a pressing need to develop biomarkers that would facilitate prediction of the progression from joint injury to joint degeneration and OA. Biomarkers would also provide an insight into disease pathogenesis and serve as a guide to the development of therapeutic interventions. MicroRNAs (miRNAs) are small (20-22 nucleotide) noncoding RNAs that have been shown to play an overarching regulatory role in normal cellular function and in many diseases. Quantification of miRNAs present in plasma and serum has been very recently shown to provide an excellent biomarker for cancer and other diseases. We have demonstrated miRNA expression by cartilage and chondrocytes and the presence of miRNAs in human and mouse serum. We have also shown that specific miRNAs have a markedly increased expression in OA cartilage suggesting a role in OA development. We propose to characterize miRNAs in the serum and joint tissues in a mouse model of OA and in the serum of patients developing OA after ACL injury to identify biomarkers of OA development. Validation of this panel of miRNA biomarkers will then be conducted in two distinct models of PTOA;a much larger group of patients with ACL injury and a sheep model of PTOA, mimicking human PTOA resulting from impact trauma. MiRNAs offer a number of advantages over traditional biomarkers for PTOA. There are fewer known miRNA species than proteins or carbohydrates, so obtaining a complete profile is relatively easy. Serum miRNAs show consistent expression between individuals and are present in a remarkably stable form. Detecting and quantifying specific miRNAs is inherently a much easier task than detecting proteins due, in part, to their small size and very similar chemical properties. A modified RT PCR assay for miRNAs is specific and extremely sensitive. MiRNAs also show a restricted tissue expression profile that provides the capacity to trace plasma or serum miRNAs to a source tissue. Furthermore analysis of serum miRNAs makes no assumption about involvement of specific joint tissues and allows for the detection of contributions from tissues other than those currently known while permitting the examination of progressive changes in joint tissues already implicated in the disease. The course of PTOA progression after knee trauma is expected to have substantial overlap with disease progression in general OA. Thus the development of biomarkers for progression of PTOA will have benefits for prediction of progression of OA pathology, provide an indication of pathways of disease pathology and serve as a guide to therapeutic intervention in PTOA as well as OA generally. The recent demonstration of miRNA biomarkers in serum or plasma together with their unique characteristics provides a new dimension in the search for biomarkers for many pathologic conditions including PTOA. The studies proposed will thoroughly assess the potential of serum miRNAs as biomarkers of PTOA using unique animal models and patient serum from to an ongoing, well-defined, human study. Osteoarthritis is a debilitating widespread disease that involves the loss of function of articular cartilage;the smooth bearing surface of our joints. For many years the research community has been trying, largely unsuccessfully, to develop biomarkers that will provide clinical guidance and enable development and assessment of treatment strategies. We propose to develop osteoarthritis biomarkers by measuring a family of newly discovered small RNA molecules present in serum and shown in other diseases to provide exciting new disease biomarkers.